Our studies have the long-term objective of understanding the mechanism(s) by which cells regulate cholesterol metabolism. These studies are related to fundamental consideration of the role of cholesterol as a key component in cytoplasmic membranes of nearly all eukaryotic cells and to clinical questions of serum cholesterol levels and atherosclerosis in man. We have isolated cell lines which are resistant to compactin (ML236B). These cells overproduce the enzyme HMG-CoA reductase which is the rate determining step in cholesterol synthesis. We will determine the mechanism of enzyme over production in these cell lines. We will isolate both cDNA and genomic copies of the gene which specifies HMG-CoA reductase. We will determine the nucleotide sequence of the gene and thus the primary structure of the enzyme. Using in vitro translation experiments we will determine if normal regulation of enzyme synthesis by physiological factors, such as cholesterol, oxygenated sterols and low density lipoproteins, is due to changes in levels of translatable reductase mRNA and later we will examine transcription of the gene directly. We will purify the enzyme in undegraded form in order to study its structure and membrane orientation. We will study how the enzyme is biosynthetically inserted into the endoplasmic reticulum.